Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL⁻¹ 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These ‘nucleotide ratios’ changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.     

Sacrificial Bioprinting of a Mammary Ductal Carcinoma Model

Cancer tissue engineering has remained challenging due to the limitations of the conventional biofabrication techniques to model the complex tumor microenvironment. Here, the utilization of a sacrificial bioprinting strategy is reported to generate the biomimetic mammary duct‐like structure within a hydrogel matrix, which is further populated with breast cancer cells, to model the genesis of ductal carcinoma and its subsequent outward invasion. This bioprinted mammary ductal carcinoma model provides a proof‐of‐concept demonstration of the value of using the sacrificial bioprinting technique for engineering biologically relevant cancer models, which may be possibly extended to other cancer types where duct‐like structures are involved.     

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.     

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPβCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.     

Changes in epithelial cells in Hirudo medicinalis during wound healing.

In the leech, Hirudo medicinalis, reepithelialization is an event which takes place early in the wound healing process, immediately after the formation of the pseudoblastema, 4-8 hr postinjury. Epithelial cells on the wound margins move into the wound, modifying their phenotypic characteristics. Cells lose their columnar shape and become flattened. Dermal junctions disrupt and tonofilaments regroup around the nucleus. Then, the epithelial cell sheet moves over the newly formed pseudoblastema by extending filopodia, formed by the cells on the edge, following the so-called "sliding model." When the wound is fully covered by the new epithelium, about 24 hr postinjury, a reorganization of the cytoskeleton occurs and the basal dermal junctions are reconstructed. Six days postinjury, the epidermal cells return to their original columnar shape.     

A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress

Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species. In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT. This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT). Expression of the QsMT cDNA in E. coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT. Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant. Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H(2)O(2) or paraquat treatments. The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed.     

Hyperghrelinemia is a common feature of Prader-Willi syndrome and pituitary stalk interruption: a pathophysiological hypothesis

BACKGROUND: Elevated plasma ghrelin levels have recently been reported in adults and children with Prader-Willi syndrome (PWS). The aim of the study is to investigate the relationship between obesity, growth hormone (GH) deficiency (GHD) and ghrelinemia in PWS and to examine whether hyperghrelinemia is specific to PWS. METHODS: We measured fasting ghrelinemia in children with PWS, idiopathic GHD (iGHD), obese children, controls and in 6 children presenting another congenital syndrome associated with GHD: pituitary stalk interruption (PSI). RESULTS: Children with PWS exhibited significantly higher ghrelin levels (995 pg/ml (801/1,099, median 1st/3rd quartile)) than iGHD (517 pg/ml (392/775)), obese (396 pg/ml (145/610)) and control (605 ng/ml (413/753)) children. Similar to PWS hyperghrelinemia was found in PSI children (1,029 pg/ml (705/1,151)), and was not modified by GH treatment. CONCLUSION: We conclude that hyperghrelinemia in PWS and PSI is not related to GH secretion. We hypothesize that a major site of ghrelin action is at the hypothalamic level and that a 'ghrelin resistance' syndrome may be present in these patients, primarily due to a hypothalamic defect. Combined alterations such as impaired serotonin receptor regulation associated with abnormal ghrelin responsiveness are probably responsible for obesity in PWS.